1. Field of the Invention
The present invention relates generally to the field of drug screening. More specifically, the present invention relates to high-throughput assays that measure virus entry and are useful for drug screening.
2. Description of the Related Art
A few entry inhibitors exist for influenza and HIV. While these inhibitors show some promise, they are far from perfect and have low efficacy. The discovery of similar but more effective drugs has been hindered by a lack of high-throughput, high signal to noise assay for screening lead compounds/drugs.
pH-dependent models such as influenza A and semliki forest virus have been used to study the mechanisms of enveloped virus entry. This is because it is possible to induce an en-masse fusion event by dropping the pH of the medium. Fluorescence dequenching or FRET assays are used to measure the kinetics of fusion in such viruses and have been used to understand the effects of mutations and anti-viral drugs (Blumenthal, R. et.al, 1987, Danieli, T., et.al., 1996). In this assay, the fluorescent probes incorporated in the virus membranes mix with and become diluted in the target cell or liposome membrane. The resulting change in the fluorescence gives a real-time measure of fusion. However, these entry assays cannot be applied easily to the pH-independent viruses since fusion events are infrequent, cannot be co-ordinated and the receptors are difficult to manipulate since they tend to be integral, multi-transmembrane span-containing proteins. In such studies, passive diffusion of the fluorophore contributes significantly to the signal and complex analysis of the data is required to observe signal due to fusion.
The need for more sensitive measurement of pH-dependent virus entry has therefore, led to the development of assays to detect cell-cell fusion, early genome replication events and assays that use recombinant viral protein-GFP fusions (Erlwein, O., et.al, 2003, McDonald, D., et.al, 2002, Spitzer, D., et.al, 2003). In cell-cell fusion assay, which is used to confirm the role of factors important in fusion, cells made to express the virus envelope proteins on the surfaces are labeled with one fluorophore and are mixed with target cells bearing receptor and second fluorophore. Fusion is measured by observing syncytia formation. Independent labeling of the cell membrane and cytoplasm provides information on membrane and cytoplasm mixing. However, syncytia formation is slow and does not correlate to infection kinetics. Additionally, in case of HIV, the chemokine receptor, Bonzo promotes syncytia but does not play any significant role in entry (Edinger, A. L., et.al. 1998, Sharron, M., et.al., 2000).
There are several assays that detect virus infection. Some assays measure infection by using the reporter gene expression in the infected cell. However, this is very complicated process. To obtain expression, a virus must penetrate the cell membrane, the core must be trafficked to the correct subcellular location, then the genome is exposed and finally the reporter is expressed. This requires for example in retroviruses, the cell to be at a specific stage and the gene expression requires at least 24 hours after contact with cells which is far removed from the initial entry event.
Other assays, which involve making virus-protein fusions to green fluorescent protein, have been useful to follow virus after it has entered the cell. The use of fluorescently labeled dUTP even permits the visualization of genomes undergoing reverse transcription (McDonald, D., et.al., 2002). However, these assays cannot be easily used to examine entry as cell bound virus cannot be differentiated from that which has just entered and for retroviruses, particle to infectious particle ratios typically exceed 10-100. This means that most viruses are either defective or trafficked to non-productive pathways within the cell.
Contents-mixing assays, which measure the release of virus contents into the cell or target vesicle, demonstrate the greatest potential for rapid measurement of virus entry. In case of retroviruses, this is most commonly done by viral DNA synthesis: transcripts can then be detected by PCR, typically around 4 hours after cell contact. However, it is not known at what point within this 4-hour window, the genome uncoating takes place and the assay is not quantitative.
A method where the enzyme β-lactamase is fused to the HIV protein, vpr (Cavrois, M., et.al., 2002) was developed recently to obtain quantitative data. Vpr is packaged into HIV particles as part of virus assembly and provided a means of targeting a marker enzyme into the particle. Caged substrate was perfused into cells to give signal. However, in practice, this assay lacked sensitivity since the detection of entry required 12 hours of cell culture for production of sufficient reaction product. To enable shorter measurement times, an MOI of more than 10-100 is required which is not physiological.
Thus, the prior art is deficient in assays that are fast, simple, physiological and sensitive to measure virus entry. The present invention fulfils this need by providing method that allows rapid and high-throughput non-radioactive detection of virus entry, has high signal to noise ratio and can detect virus entry into cells within 30 minutes after virus-cell contact.